Post on 18-Mar-2021
Héctor Peinado Selgas, PhD.Head, Microenvironment and Metastasis GroupSpanish National Cancer Research Centre (CNIO)
Critical steps for circulating exosomes analyses
Where are we? Exosomes in liquid biopsy
Exosomes in liquid biopsy
Exosomes
Adapted from Int. J. Mol. Sci. 2017, 18(2), 264;
Use of exosomes to detect tumor mutation heterogeneity
Exosomes
Adapted from Amirouchene-Angelozzi N, Cancer Discov. 2017
Limitations: Extracellular vesicles = exosomes
Oncosomes
CytoplastExomere
Adapted from Cell Mol Life Sci. 2011 Aug;68(16):2667-88.
Exosomes in liquid biopsy = tumor exosomes
Proteins (receptors, oncoproteins…)RNA (mRNA, smallRNA, rRNA…)DNA (ssDNA, dsDNA, retrotransposons)Lipids (sphingomyelin, ceramides…)Sugars (glycosilation)Metabolites
Representation of tumor-derived cargo <10%
Potential tumor exosomal markers for diagnosis (so far)
Sometimes… I see tumor-derived exosomes
Transl Cancer Res 2018;7(Suppl 2):S226-S242
Use of circulating exosomes in melanoma
Retrospective series of plasma from Stage III melanoma patient
(4 years followed up)
– POD – progression of disease , NED – no evidence of disease
Peinado et al. Nature Medicine. (18):883–891 (2012)Protocol: UltracentrifugationDetection: WBOrigin: EDTA-Frozen
Use of circulating exosomes in melanoma
Alvaro Gonzalez, Salvador Martin-Algarra, Clínica Navarra
Protocol: ExoquickDetection: ELISAOrigin: Serum-Frozen
Caution: in need of protocol standardization
Collection: Fresh, Frozen
Material: Plasma, Serum, Other
Protocol: Ultracentrifugation, Filtration, SEC, Kit
Measurement: Nanosight, Zeta, …
Detection: WB, ELISA, Mesoscale
Material: Protein, miRNA, DNA
Or at least… specification
2019 – Between pre-clinical and clinical application
Problems we face:
• Tumor exosomes represent a MINORITY of circulating exosomes –
unbiased analysis NOT RECOMENDABLE
• Results depend on the methods – NO STANDARD (e.g fresh/frozen, tubes,
ELISA, WB …)
• Studies are not properly design (n, treatment, doses, material, timing)
• Use in diagnosis is feasible – MARKER NEEDED
• Application in response to therapy or specific mutation
Detection of DNA in circulating exosomes
Thakur et. al. Cell Research (2014) 24:766–769.
WGS of human colon cancer cell line
ExoDNA represents the whole genome DNA
HCT116 ExoDNA
HCT116 gDNA
Regions below 10 Kb are the most represented6951 regions represented 10Kb, 5 regions represented 100Kb
WGS of B16-F10 cancer cell line
gDNA
ExoDNA
Thakur, Peinado, Lyden Unpublished
ExoDNA reflects the mutational status of parental cells
Non-Small-Cell Lung Cancer cell exoDNA
Melanoma exoDNA
WT: Sk-Mel103, 146 & 147; V600E: Sk-Mel28, 133, 192 & 267“V”, WT allele; “E”, mutant allele.
Thakur et. al. Cell Research (2014) 24:766–769.
Improving detection– ExoNAs vs cfDNA
Similar Sensibility
Overall survival (OS) per mutation allelic frequency (MAF) of KRAS, BRAF, or EGFR mutations in plasma
Clin Cancer Res. 2018 Jan 1;24(1):181-188.
Dr. Hector Peinado´s GroupMicroenvironment and Metastasis Group
Molecular Oncology Program, CNIOMadrid, Spain
Susana García, PhD. – Staff ScientistMarta Hergueta, PhD – PostdocClaudia Savini, PhD - PostdocLaura Nogues, PhD – Postdoc Ana Isabel Amor, Severo Ochoa – PhD StudentLucia Robado de Lope, La Caixa – PhD StudentTeresa Gonzalez Muñoz – FPU PhD StudentAlberto Hernandez – PhD StudentMarina Mazariegos – TechSara Sanchez-Redondo, TechAlicia Teijeira, Ms. Student.
Collaborators:
Jasminka Boskovic, Mercedes Robledo, Javier Muñoz - CNIO
Pablo L. Ortiz-Romero, José Luis Rodríguez-Peralto – H12O,
Madrid, Spain
Piotr Rutkowsky, Marie Curie Oncology Center, Poland
Laura Santambrogio (Albert Einstein) Mary Sue Brady (MSKCC).
USA
Johan Skog, Mikkel Noerholm – Exosome Diagnostics
Former Members:Alberto Benito Martin, PhD – WCM, NY, USACristina Merino, Tech. - GSK, MadridAngela Di Giannatale – IRCCS Bambino Gesù, Rome